In silico predictions of miRNA-mRNA interactions
For the analysis of possible miRNAs that interact with Aire mRNA, a search was performed in public databases to predict miRNA-mRNA interactions using the MirWalk platform, which is available online at (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/miRretsys-self.html). The interaction of miR-155 with the 3’UTR of Aire mRNA was predicted using the PITA algorithm, which is available at https://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html.
The thermodynamic stability of the molecular interaction was then validated using the RNAhybrid algorithm (https://bibiserv2.cebitec.uni-bielefeld.de/rnahybrid), which analyzes the secondary structure of the interaction and determines the most favorable hybridization site between a determined miRNA and the exact sequence of the 3’UTR of its target mRNA. We only selected interactions with Gibbs free energy ≤ − 20 kcal/mol, which are the most stable.
The algorithms of the MirWalk platform were used to predict other mRNAs, in addition to Aire mRNA, which could be targeted by miR-155.
Validation of miRNA-mRNA interactions through luciferase reporter gene assay (LRGA)
In this study, we used an oligonucleotide encompassing the 3’UTR of Aire mRNA synthesized through GBlocks technology by Integrated DNA Technologies (IDT, Coralville, IA, USA) as follows (the miRNA binding site is underlined), WT: /5PHOS/AAATGACAGGTGGCCCAGGAAGGGGTGGGCAGAGGCCTGGGCTGATTAGGACCACACAGCATTGGCTCCCTCCCCACCCAGCCCCATCGGATGAGGCACTCTGTTCTGAGAGGCCTGGGCTGATTAGGACCAAGAGCTGGCAGGTTCTGGCCTGCTGGAGGCCTGGGCTGATTAGGACCAACTCAGCTTGCAGATGGCCCTGATCTTTGTAGAGATGCAAGGCCACCCCATATCCTGGAATTAAAGTCACTCTAGATTCTATGTACTTTGAGGTA and MUT: /5PHOS/AAATGACAGATGACCCGTAAGGG TGGACAGCACAGCATTGGCTCCCTCCCCACCCAGCCCCATCGGATGAGGCACTCTGTTCTGAGATGACTATCTTACTGCACTCGTAGATCTGACAGGTCTGACCTGCTGTACTCGCTTGCAGATGGCCCTGATCTTTGTAGAGATGCAAGGCCACCCCATATCCTGGAATTAAAGTCATCTAGACTTCTATGTACTTTGA GGTA.
The oligonucleotide was cloned into the polycloning site of the pmirGLO vector (Promega Corporation) between the XhoI/XbaI restriction sites, resulting in the miRNA target region assuming the correct 5′ to 3′ orientation immediately downstream of the luciferase gene.
For the selected target, we introduced mutations along the 3’UTR sequence. The constructs, named “pMIR-Aire-wt-3’utr” to indicate the inclusion of the wild-type sequence and “pMIR-Aire-mut-3’utr” to indicate the inclusion of the mutant sequence, were selected by colony polymerase chain reaction (PCR) using a pair of primers flanking the vector polycloning site. We used Escherichia coli DH5α for cloning.
For the LRGA, 0.2 μg of each pmirGLO construct was transfected into human HEK-293 T cells  (6 × 104 cells/well) with 1.6 pmol of miR-155 or scrambled miRNA mimic negative control (both from Thermo Scientific Dharmacon, Waltham, MA) in a 96-well plate. Transfections were performed using the Attractene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The transfected cells were incubated at 37 °C in a 5% CO2 incubator; 24 h after transfection, the cells were lysed in Passive Lysis Buffer. The firefly and Renilla luciferase activities were measured in a Synergy 2 luminometer (BioTek Instruments, Inc., Winooski, VT) using the Dual-Luciferase Reporter System (Promega Corporation) according to the manufacturer’s instructions.
The LRGA results are presented as the standard error of the mean (SEM). The differences were evaluated by one-way ANOVA followed by Student’s t-test (two groups). P < 0.05 was considered to be statistically significant.
Our laboratory has national biosafety approval (National Technical Committee for Biosafety, Brasilia, Brazil, CTNBio Permit No. 0040/98).
mTEC 3.10 cell line, miR-155-5p mimic transfection and total RNA extraction
We used the murine mTEC 3.10 cell line previously characterized as Aire+, EpCAM+, Ly51−, UEA1+ . These cells were isolated by other researchers [51, 52] from a C57Bl/6 mouse (Mus musculus) thymus and were shared with us.
For the transfection of mTEC 3.10 cells with the miR-155-5p mimic, we used an oligonucleotide coupled to ZEN [N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine] stabilizing molecule and synthesized by IDT whose sequence was 5’ZEN-UUAAUGCUAAUUGUGAUAGGGG-ZEN3’. The cells were transfected using Lipofectin Reagent from Gibco-BRL Life Technologies (Van Allen Way Carlsbad, CA, USA) following the manufacturer’s protocol. The mTEC 3.10 cells were grown in RPMI medium and 10% inactivated fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere in T25 polystyrene flasks until they reached 50% confluence.
The miR-155-5p mimic-lipofectamine mixture was prepared by mixing 1 μL of 10 μM miR-155 mimic with 500 μL of Opti-MEM I Reduced Serum Medium Gibco (Paisley, Scotland, UK). Next, 10 μl of Lipofectin Reagent was added. This mixture was vigorously stirred for 10 seconds and then incubated for 10 minutes at room temperature.
The miR-155-5p mimic-lipofectamine complex was added to the culture flasks in a total volume of 5 ml per bottle, resulting in a concentration of 2 nM miR155 mimic. For the control, mTEC 3.10 cells were cultured under similar conditions, except that the miR-155 mimic was added alone. The cells were cultured for different periods (12, 24, and 48 h posttransfection). Independent experiments and controls were repeated three times.
Total RNA was extracted from control or transfected cells using the mirVana miRNA Isolation Kit (Ambion, Grand Island, NY, USA) according to the manufacturer’s instructions. The integrity of the RNA samples was evaluated through microfluidic gel electrophoresis in RNA 6000 nanochips and in an Agilent 2100 Bioanalyzer device (Agilent Technologies, Santa Clara, CA, USA). We used only RNA preparations with an RNA integrity number (RIN) ≥ 7.5 that were free of phenol (A260/A220 approx. 0.7) and free of protein (A260/A280 approx. 2.0).
Reverse transcription for cDNA synthesis
mRNA reverse transcription reactions were performed with components of the Applied Biosystems (Foster City, CA, USA) High Capacity cDNA Reverse Transcription Kit following the manufacturer’s protocol. For these reactions, 500 ng of total RNA was used. For the reverse transcription of miR-155, the TaqMan MicroRNA Reverse Transcription Kit (Foster City, CA) was used following the manufacturer’s protocol. For these reactions, 50 ng of total RNA was used.
Real-time quantitative PCR (RT-qPCR)
The expression levels of Aire mRNA and miR-155 were assessed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). For these reactions, TaqMan Gene Expression Assay probes specific for Aire, Gapdh (endogenous control for mRNA), miRNA-155, and snoRNA-202 (endogenous control for miRNA) were used. The TaqMan Fast Advanced Master Mix kit (Applied Biosystems) was used following the manufacturer’s protocol.
The expression level of miR-155 was normalized to that of snoRNA202, which is commonly used as an endogenous control for miRNA expression analysis.
The expression levels of the Aire gene were normalized to those of the constitutive gene Gapdh (ENSMUSG00000057666), which is commonly used as a reference.
The relative quantification of gene expression was performed using the 2-ΔΔCT method . For the statistical analysis of the data, Student’s t-test and one-way ANOVA were performed using GraphPad Prism statistical software (www.scolary.com/tools/graphpad-prism) according to the manufacturer’s instructions.
Extraction and quantification of total proteins from mTEC 3.10 cells
Approximately 5 × 105 cells were seeded in T75 culture polystyrene flasks containing 10 mL of RPMI 1640 medium and 10% fetal bovine serum (FBS). The cells were transfected with the miR-155-5p mimic and cultured for 24, 36, or 48 h. The cells were detached from the culture flasks by the conventional trypsin treatment. The cells were washed three times with 1x PBS and centrifuged at 1000 x g for 5 minutes. Then, 300 μL of modified RIPA buffer [50 mM TrisCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% protease inhibitor cocktail (Sigma-Aldrich, Darmstadt, Hesse, Germany)], and the cells were lysed using a Polytron homogenizer. The homogenate was then centrifuged at 10,000 x g for 20 minutes at 4 °C. The total protein content of the supernatant was determined by using conventional Bradford reagent (Bio Rad, Hercules, CA, USA).
Western blot analysis of the AIRE protein
The electrophoresis of total proteins in denaturing polyacrylamide gels (SDS-PAGE) was performed using the discontinuous buffer system described by Laemmli and Favre . Polyacrylamide mini gels (10 × 8 cm; 0.075 cm thick) were used. The concentration of the stacking gel was 4%, and the concentration of the separation gel was 10%. The protein samples were prepared for electrophoresis at a ratio of 3:1 (vol/vol) of sample to 4x concentrated sample buffer (240 mM Tris-HCl pH 6.8; 8% SDS; 40% glycerol; 20% 2-mercaptoethanol and bromophenol blue). The samples were heated for 5 minutes at 95 °C in a heat block. The electrode buffer contained 25 mM Tris-HCl, pH 8.3; 192 mM glycine; and 0.1% SDS. The protein molecular weight standard Precision Plus Protein™ Dual Color Standards (BioRad, Hercules, CA, USA) was used.
The protein bands, once separated, were transferred from the gel to a PVDF membrane (BioRad, Hercules, CA, USA) using 25 mM Tris-HCl, 190 mM glycine, 20% methanol, and 1% SDS as transfer buffer at 80 V for 2 hours. For this step, we used the BioRad Mini Trans-Blot Cell device (Hercules, CA, USA). After the transfer, the membrane was stained with 0.2% Ponceau S in 3% trichloroacetic acid for 5 minutes to evaluate the transfer efficiency. Then, the membrane was destained with deionized water. The membrane was blocked with skim milk (MOLICO) diluted to 5% in TBS-Tween (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% Tween-20) for 1 hour at room temperature. After blocking, the membrane was incubated with the polyclonal anti-Aire D17 (goat antibody) primary antibody from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA), which was diluted in TBS-T (1:500), for 18 h, followed by three washes in TBS-T for 5 minutes each.
The membrane was incubated with the anti-goat IgG secondary antibody conjugated to peroxidase (horseradish-peroxidase - HRP), which was diluted 1:10,000 in TBS-T, for 1 hour. After incubation, the membrane was washed in TBS-T, as before. The development was performed in an ImageQuant LAS 500 Ge Life Sciences apparatus (Piscataway, NJ, USA) using the appropriate substrate of peroxidase (Luminata Forte, Merck Millipore). The reaction was conducted in the dark, and we exposed the membrane for different time intervals. The conversion of the intensity of the protein bands in the images into numerical values was performed with the aid of the ImageJ 1.49 program (http://imagej.nih.gov/ij/index.html). The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) protein was used as an internal reference in the analyses. Thus, the membrane was also incubated with the polyclonal (rabbit) anti-GAPDH primary antibody from Cell Signaling Technology (Beverly, MA, USA). In that case, a secondary anti-rabbit IgG antibody conjugated to peroxidase was used. The immunostaining procedure was the same as that used for Aire.
Immunolocalization of the AIRE protein
To assess the intracellular localization of the AIRE protein, we used the immunofluorescence of mTEC 3.10 cells (control or transfected with the miR-155 mimic). For this experiment, we used the anti-AIRE D17 primary antibody from Santa Cruz Biotechnology (Dallas, Texas, USA). For this assay, 3 × 104 mTECs (3.10) were plated on 13-mm2 glass coverslips placed in a 24-well plate. After transfection with the miR-155-5p mimic, the culture medium was removed from the wells, and the coverslips were washed five times with PBS.
The cells were fixed on the coverslips with 300 μL of 4% paraformaldehyde in PBS for 15 minutes. The cells were then subjected to three washes with PBS for 5 minutes each and then permeabilized with PBS containing 0.5% Triton X-100 for 5 minutes.
After three more washes with PBS (5 minutes each), the cells were blocked for 1 hour with 2% BSA in PBS. A diluted (1:50 in PBS containing 1% BSA) primary antibody (anti-AIRE D17) was added, followed by incubation at room temperature for 1 hour. The cells were washed three times with PBS (10 minutes each). Then, the Novex TM mouse anti-goat IgG (H + L) rhodamine red-conjugated secondary antibody (Life Technologies Corporation, Carlsbad, CA, USA), which was diluted 1:500 in PBS containing 1% BSA, was added and incubated for 1 hour. The coverslips were washed with PBS (3 × 10 minutes).
To visualize the cytoplasmic region, the actin filaments were labelled with phalloidin conjugated to AlexaFluor 488 (Life Technologies; Eugene, Oregon, USA) according to the manufacturer’s instructions. The coverslips were mounted with ProlongGold Antifade Mountant (Life TechnologiesTM - Eugene, Oregon, USA) containing DAPI (to visualize the nuclei). The coverslips were visualized and photographed using an ApoTome Zeiss fluorescence microscope (Oberkochen, Germany).
Microarray hybridizations and data analysis
This study followed a protocol that was previously established in our laboratory for microarray hybridizations . Briefly, total RNA preparations were used to synthesize dscDNAs from mRNAs, and then, cyanine 3 (Cy3)-CTP-labeled complementary amplified RNAs (cRNAs) were synthesized. For these reactions, we used the Agilent Linear Amplification kit (Agilent Technologies) according to the instructions of the manufacturer. The (Cy3)-cRNA samples were hybridized to Agilent mouse 4 × 44 K-format oligonucleotide microarrays (Agilent Technologies). The slides were washed according to the manufacturer’s instructions and scanned with an Agilent DNA microarray scanner.
The hybridization signals from the scanned microarray slides were extracted using Agilent Feature Extraction software, version 10.7.1.1. The expression profiles of independent samples of the control or miR-155-5p mimic-transfected mTEC 3.10 cells were analyzed by comparing the microarray hybridization signals of the respective samples. The numerical and quantitative microarray data were normalized to the 75th percentile and analyzed in the R statistical environment (version 3.3.1) (https://www.r-project.org). In the data preprocessing phase, we used the arrayQualityMetrics  and Agi4x44PreProcess  tools, which have algorithms that allow the qualitative evaluation of arrays in addition to the correction of the background and normalization of the data.
For the analysis of differentially expressed mRNAs, we used the functions of the Limma package , which applies a linear model to the gene-wise statistical analysis. For an analysis of multiple tests, empirical Bayes and the Benjamini-Hochberg correction were applied.
In this work, we considered a p-value ≥0.05 with correction by FDR (Benjamini-Hochberg) and fold-change ≥1.5 to indicate differentially expressed mRNAs.
The differentially expressed transcripts were then subjected to hierarchical clustering, and heat maps were constructed to evaluate the expression pattern of the mRNAs. The Euclidean distance and the complete linkage method were used to group the samples and the RNAs. The functional enrichment of the differentially expressed (DE) mRNAs was done through the Database for Annotation, Visualization, and Integrated Discovery (DAVID) annotation tool (https://david.ncifcrf.gov/). This tool was used for the identification of the main biological processes and pathways represented by DE mRNAs. A functional category was considered significant if it had at least three mRNAs and a score of p < 0.005 with Benjamini-Hochberg correction. Tissue-restricted antigen (TRA) mRNAs were defined as Samson et al. . Briefly, an mRNA encoding a TRA was identified according to its higher expression in up to five different tissues concerning the expression mean of the whole set of the differentially expressed mRNAs.
Thymocyte migration assay
For the separation of thymocytes, female C57BL/6 mice aged 5-6 weeks old were killed in a CO2 chamber, and the thymuses were quickly removed by thoracic surgery. The organs were fragmented with the aid of two surgical forceps in RPMI 1640 culture medium in a Petri dish. The thymocytes were separated through a 10 μm pore size nylon membrane (Sefar Inc. Depew, NY, USA). The thymocyte suspension was centrifuged for 5 minutes at 1000 x g, and the cells were washed twice with PBS and then resuspended in RPMI 1640 medium.
For the thymocyte migration assay, we used a protocol previously published  with modifications as follow. Transwell chambers and 6.5 mm inserts with a 0.5 μm in diameter polycarbonate membrane (Corning Inc. Corning, NY, USA) were used. The inserts were placed in 24-well plates and incubated in PBS for 1 hour at 37 °C and then blocked in PBS containing 10 μg BSA/mL for 1 minute.
The inserts were then transferred to wells containing conditioned culture medium, i.e., medium collected from mTEC 3.10 control cell culture or mTEC 3.10 transfected with miR-155 mimic (cell cultures were grown for 24 hours). The conditioned medium was filtered through 0.45 μ filters before use. 100 μL of conditioned medium from the control cultures or the transfected cultures were placed in each well. A suspension containing 2.5 × 106 thymocytes was then deposited on each of the inserts. The inserts were then placed inside the wells and incubated at 37 °C in an atmosphere with 5% CO2 for 3 hours to allow the migration of thymocytes.
The inserts were then placed in wells containing 70% ethanol for 10 minutes to fix the thymocytes in passing through the pores. The outer surface of each insert was stained with Giemsa for 10 minutes and finally washed in deionized water to remove excess dye and then left to dry in room air. The inserts were visualized and photographed in a stereomicroscope model Stemi 508 Zeiss (Oberkochen, Germany).
The images were analyzed considering the average pixel of the migrated thymocytes (pixels/area). Thymocyte migration ranged from zero (no migration) to 255 pixels, which was the maximum pixel value for migrated and stained thymocytes. Migration values were compared between thymocytes that migrated in conditioned culture medium versus conditioned culture medium of cells transfected with miR-155 mimic.